Existence of uncoupling protein-2 antigen in isolated mitochondria from various tissues.

Antibodies against Escherichia coli-expressed uncoupling protein-2 (UCP2) and uncoupling protein-3 (UCP3) were raised by operating the blotted proteins into the spleen of minipigs. The antisera reacted more intensively with the recombinant UCP2 and UCP3 than with uncoupling protein-1 (UCP1) isolated from brown adipose tissue. Moreover, anti-UCP2 and cross-reacting anti-UCP3 antibodies identified the presence of the UCP2/3 antigen in isolated mitochondria from rat heart, rat kidney, rat brain, rabbit epididymal white adipose tissue, hamster brown adipose tissue, and rabbit skeletal muscle. It has been concluded that UCP2 is expressed in these tissues (UCP3 in skeletal muscle); however their existence in mitochondria had not previously been demonstrated.     

SSR182289A, a selective and potent orally active thrombin inhibitor

SSR182289A 1 is the result of a rational optimisation process leading to an orally active thrombin inhibitor. The structure incorporates an original 2-(acetylamino)-[1,1'-biphenyl]-3-sulfonyl N-terminal motif, a central l-Arg surrogate carrying a weakly basic 3-amino-pyridine, and an unusual 4-difluoropiperidine at the C-terminus. Its synthesis is convergent and palladium catalysis has been employed for the construction of the key C-C bonds: Suzuki coupling for the bis-aryl fragment and Sonogashira reaction for the delta- bond of the central amino-acid chain. The compound is a potent inhibitor of thrombin's activities in vitro and demonstrates potent oral anti-thrombotic potencies in three rat models of thrombosis. The observed in vitro potency could be rationalized through the examination of the interactions within the SSR182289A 1 - thrombin crystal structure. SSR182289A 1, has been therefore selected for further development.     

DNA polymerase beta overexpression stimulates the Rad51-dependent homologous recombination in mammalian cells

Overexpression of DNA polymerase β (polβ), an error-prone DNA repair enzyme, has been shown to result in mutagenesis, aneuploidy and tumorigenesis. To further investigate the molecular basis leading to cancer-associated genetic changes, we examined whether the DNA polβ could affect homologous recombination (HR). Using mammalian cells carrying an intrachromosomal recombination marker we showed that the DNA polβ overexpression increased the HR mostly by enhancing gene conversion. Concomitantly, we observed the generation of DNA strand breaks as well as a DNA polβ-dependent formation of Rad51 foci. The stimulation of HR was abolished by the coexpression of a dominant negative form of Rad51, suggesting that the Rad51 was involved in the increased HR events. The expression of different DNA polβ mutants lacking polymerase activity did not result in HR stimulation, indicating that the DNA synthesis activity of DNA polβ was related to this phenotype. These results provide new insights into the molecular mechanisms of the genetic instability observed in DNA polβ overexpressing tumour cells.     

Distinct requirements for IFNs and STAT1 in NK cell function

NK cell functions were examined in mice with a targeted mutation of the STAT1 gene, an essential mediator of IFN signaling. Mice deficient in STAT1 displayed impaired basal NK cytolytic activity in vitro and were unable to reject transplanted tumors in vivo, despite the presence of normal numbers of NK cells. IL-12 enhanced NK-mediated cytolysis, but poly(I:C) did not, and a similar phenotype occurred in mice lacking IFNalpha receptors. Molecules involved in activation and lytic function of NK cells (granzyme A, granzyme B, perforin, DAP10, and DAP12) were expressed at comparable levels in both wild-type and STAT1(-/-) mice, and serine esterase activity necessary for CTL function was normal, showing that the lytic machinery was intact. NK cells with normal cytolytic activity could be derived from STAT1(-/-) bone marrow progenitors in response to IL-15 in vitro, and enhanced NK lytic activity and normal levels of IFN-gamma were produced in response to IL-12 treatment in vivo. Despite these normal responses to cytokines, STAT1(-/-) mice could not reject the NK-sensitive tumor RMA-S, even following IL-12 treatment in vivo. Whereas in vitro NK cytolysis was also reduced in mice lacking both type I and type II IFN receptors, these mice resisted tumor challenge. These results demonstrate that both IFN-alpha and IFN-gamma are required to maintain NK cell function and define a STAT1-dependent but partially IFN-independent pathway required for NK-mediated antitumor activity.     

Function of inflammatory cells and neoral cyclosporin-A in heart transplant-associated coronary vasculopathy

The role of Sandimmun Neoral (S-n) and the immune response in transplant-associated coronary vasculopathy (TACV) was evaluated in a Lewis (Lew)-to-Fischer-344 (F344) rat abdominal heterotopic heart transplant model. Some of the transplant recipients were treated with S-n (5mg/kg/day) for 14 days post-transplant, or until sacrifice. Grafts were subjected to immunohistochemical (ED1, CD4, CD8 and alpha-actin+ cells) analysis from day 7 to 100 post-transplant. Singenic controls did not develop TACV, irrespective of whether they had received the drug or not. TACV was detected in Lew-F344 transplants regardless of S-n administration with participation of ED1+, CD8+ and alpha-actin+ cells, although its incidence was lower in animals receiving prolonged S-n treatment. In this model, accelerated arteriosclerosis of the graft appeared to be related more to the rejection effect than to the action of the immunosuppressive agent.     

Functional Mapping of Human Dynamin-1-Like GTPase Domain Based on X-ray Structure Analyses

Human dynamin-1-like protein (DNM1L) is a GTP-driven molecular machine that segregates mitochondria and peroxisomes. To obtain insights into its catalytic mechanism, we determined crystal structures of a construct comprising the GTPase domain and the bundle signaling element (BSE) in the nucleotide-free and GTP-analogue-bound states. The GTPase domain of DNM1L is structurally related to that of dynamin and binds the nucleotide 5′-Guanylyl-imidodiphosphate (GMP-PNP) via five highly conserved motifs, whereas the BSE folds into a pocket at the opposite side. Based on these structures, the GTPase center was systematically mapped by alanine mutagenesis and kinetic measurements. Thus, residues essential for the GTPase reaction were characterized, among them Lys38, Ser39 and Ser40 in the phosphate binding loop, Thr59 from switch I, Asp146 and Gly149 from switch II, Lys216 and Asp218 in the G4 element, as well as Asn246 in the G5 element. Also, mutated Glu81 and Glu82 in the unique 16-residue insertion of DNM1L influence the activity significantly. Mutations of Gln34, Ser35, and Asp190 in the predicted assembly interface interfered with dimerization of the GTPase domain induced by a transition state analogue and led to a loss of the lipid-stimulated GTPase activity. Our data point to related catalytic mechanisms of DNM1L and dynamin involving dimerization of their GTPase domains.     

Soporte nutricional con nutrición parenteral. Evolución y complicaciones asociadas

IntroductionParenteral nutrition (PN) is an integral part of medical management of patients who do not have a functioning or accessible gastrointestinal tract. This paper discusses the clinical characteristics of patients receiving PN in a 420-bed hospital from 2009 to 2011. In addition, nutritional parameters were assessed at the start and end of PN and associated complications were analyzed.Material and methodsretrospective, observational study of PN episodes in adults conducted at the Nutrition Unit of Hospital Universitario de Guadalajara. Variables collected included epidemiological and clinical data, number and type of routes used, anthropometric data, analytical data, number of days on PN, reason for withdrawal, caloric provision, prevalence of phlebitis, metabolic complications (hypertriglyceridemia, abnormal liver function tests, hyperglycemia, and refeeding syndrome), and prevalence of bacteremia associated with central venous catheter (BAC).ResultsThere were 312 episodes of PN. The immediate indication was postoperative ileus in 53.8% of the episodes. There was a statistically significant improvement in all analytical parameters assessed (albumin, prealbumin, retinol binding protein, transferrin, cholesterol, and lymphocytes). Caloric provision (kcal per kg) was 25.1 ± 6.6. No metabolic complication occurred in 16.3% of the episodes, and hyperglycemia was the most common complication (79.8%). There were 10 cases of phlebitis (32.2%) and 30 episodes of BAC (8.7%). Bacteriemia rate was 8.1 per 1000 days of PN.DiscussionAlthough PN is an effective nutritional support technique, it is associated with complications of varying severity. Use of PN should therefore comply with the instructions accepted in the main clinical practice guidelines and requires careful monitoring by experienced professionals.